The sequencing of deoxyribonucleic acid (DNA) is a fundamental tool of modern biology and is conventionally carried out in various ways, commonly by processes which separate DNA segments by electrophoresis. Such sequencing techniques can be used to determine which genes are active and which are inactive, either in specific tissues, such as cancers, or more generally in individuals exhibiting genetically influenced diseases. The results of such investigations can allow identification of the proteins that are good targets for new drugs or identification of appropriate genetic alterations that may be effective in genetic therapy. Other applications lie in fields such as soil ecology or pathology where it would be desirable to be able to isolate DNA from any soil or tissue sample and use probes from ribosomal DNA sequences from all known microbes to identify the microbes present in the sample.
The conventional sequencing of DNA using electrophoresis is typically laborious and time consuming. Various alternatives to conventional DNA sequencing have been proposed. One such alternative approach utilizes an array of oligonucleotide probes synthesized by photolithographic techniques. Cyclic DNA chain growth is performed by consecutive attachment of a base to a preexisting strand on a solid support such as a glass or silicon substrate. The surface of the solid support or substrate, as modified with photolabile protecting groups, is illuminated through a photolithographic mask, yielding reactive hydroxyl groups in the illuminated regions. A 3′ activated deoxynucleoside, protected at the 5′ hydroxyl with a photolabile group, is then provided to the surface such that coupling occurs at sites that had been exposed to light. Following oxidation, for molecular bond stabilization, and capping, to prevent subsequent unwanted (photo) chemical reactions, the substrate is rinsed and the surface is illuminated through a second mask to expose additional hydroxyl groups for coupling. A second 5′ protected activated deoxynucleoside base is presented to the surface. The selective photodeprotection and coupling cycles are repeated to build up levels of bases until the desired set of probes is obtained. A variation of this process uses polymeric semiconductor photoresists, which are selectively patterned by photolithographic techniques, rather than using photolabile 5′ protecting groups.
It may be possible to generate high density miniaturized arrays of oligonucleotide probes using such photolithographic techniques wherein the sequence of the oligonucleotide probe at each site in the array is known. These probes can then be used to search for complementary sequences on a target strand of DNA, with detection of the target that has hybridized to particular probes accomplished by the use of fluorescent markers coupled to the targets and inspection by an appropriate fluorescence scanning microscope.
A disadvantage of the approaches for light directed DNA synthesis that employ photolithographic masks is that four different lithographic masks are needed for each monomeric base, and the total number of different masks required are thus four times the length of the DNA probe sequences to be synthesized. The high cost of producing the many precision photolithographic masks that are required, and the multiple processing steps required for repositioning and alignment of the masks for every exposure, contribute to relatively high costs and lengthy processing times.
To overcome the limitations associated with using photolithographic masks for DNA synthesis, a method and apparatus for the synthesis of arrays of DNA probe sequences, polypeptides, and the like without photolithographic masks by using a dynamic mask image produced by an array of switchable optical elements, such as a two-dimensional array of electronically addressable micromirrors, has been developed. Each of the micromirrors can be selectively switched between one of at least two separate positions so as to contribute light to the mask image in a first position, and to deflect the light to an absorber in a second position. Projection optics receive the light reflected from the optical array and produce an image of the mirrors onto a flow cell or onto an array where the nucleotide addition reactions are conducted.
The image of the micromirrors projected onto the reaction site is generally that of a set of rectangular “pixels” corresponding to the outline of the micromirrors. Each pixel is either dark or brightly illuminated depending on the position of the corresponding mirror. Synthesis of the DNA probes, which occurs within the area of the imaged pixels, must be separated so that when the probes are scanned with an optical scanner, such as the fluorescence scanning microscope, to detect hybridization with sample DNA, the particular pixel where hybridization occurs can be unambiguously identified. The pixels are separated by dark “lanes” or “streets” corresponding to the spaces between the movable mirrors. These lanes, if clearly resolved in the image of the micromirrors at the reaction site, assist in distinguishing and identifying each pixel.
Various effects occurring during the process of light directed synthesis of a DNA microarray in the manner described can adversely affect the quality (purity) of the DNA sequences produced. The acquisition of DNA oligonucleotides from such DNA microarrays is a new expansion of the use of DNA microarrays from “gene expression” to “gene assembly”. DNA microarray quality (purity) thus becomes increasingly important, due to the strong dependency of DNA assembly success on purity of the input (construction) DNA oligonucleotides.
Various methods for error reduction in light directed DNA microarray synthesis can be categorized roughly into two approaches, error reduction (removal) during synthesis and error correction after synthesis. An example of error reduction during synthesis is the use of capping during the synthesis process. During the base coupling step of the DNA synthesis process certain coupling sites that have been exposed to light and thus de-protected for coupling may remain uncoupled. After the coupling step, a capping reagent may be used to disable these sites permanently, to prevent unwanted (photo) chemical reactions at these sites later in the synthesis process that might otherwise result in the presence of unintended DNA sequences in the microarry. Examples of error correction after synthesis include elution processes and pre/post assembly processes.
In theory, light directed DNA synthesis occurs only at reactive regions of the substrate that are illuminated intentionally via the mask pattern. However, light scattering, flair, and diffraction are optical properties that are always present at exposure of the substrate upon which the DNA is to be synthesized. For example, diffraction effects form localized patterns depending on an array configuration. There is a gradual transition of exposure doses at the pixel edges of the mask pattern. Thus, areas of the substrate that are intended to remain inactive and that are near the edges of areas that are being intentionally exposed also will receive some light exposure due to diffraction. Scattering and flair result in a more uniform distribution of sequence errors. In general, the optical properties of light scattering, flair, and diffraction may result in exposure of areas of the substrate that are intended to remain inactive, i.e., where the exposure nominal value should be zero. This can result in deprotection of sites in unintended locations on the substrate and the possible synthesis of unintended DNA sequences at those locations, thereby adversely affecting the quality of the DNA synthesis. This effect of unwanted light exposure is accumulated, since the synthesis process requires repetitive cycles of optical masking and exposure. Thus, DNA synthesis errors due to these optical effects increase with the synthesis of longer DNA sequences.
Reduction of the adverse effects of light scattering, flair, and diffraction in light directed DNA synthesis can improve significantly the quality (purity) of the resulting DNA microarray. However, light scattering, flair and diffraction effects cannot be entirely eliminated in principle. Error reduction in light directed DNA synthesis by reducing such effects directly may be achieved only at great effort and expense by complicating optical exposure systems.
What is desired, therefore, is a system and method for light directed DNA synthesis wherein the errors in DNA synthesis caused by light scattering, flair, and diffraction are reduced significantly in-situ. Preferably such error reduction is achieved by eliminating to the greatest extent possible the effects of light scattering, flair and diffraction on the synthesis process without requiring the use of expensive and complicated optical systems.